Monitoring a physiological analytes

ABSTRACT

Methods and devices are provided for measuring the concentration of target chemical analytes present in a biological system. Device configuration and/or measurement techniques are employed in order to reduce the effect of interfering species on sensor sensitivity. One important application of the invention involves a method and device for monitoring blood glucose values.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/437,784, filed 14 May 2003, now U.S. Pat. No. 6,850,790, which is acontinuation of U.S. patent application Ser. No. 10/026,086 filed 20Dec. 2001, now U.S. Pat. No. 6,594,514, which is a continuation of U.S.patent application Ser. No. 09/639,913, filed 16 Aug. 2000, now U.S.Pat. No. 6,356,776, which is a continuation of U.S. patent applicationSer. No. 09/309,727, 11 May 1999, now U.S. Pat. No. 6,144,869, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/085,373,filed 13 May 1998, all which applications are incorporated herein byreferenced in their entireties.

FIELD OF THE INVENTION

The invention relates generally to methods for measuring theconcentration of target chemical analytes present in a biologicalsystem. One important application of the invention involves a method formonitoring blood glucose concentrations.

BACKGROUND OF THE INVENTION

A number of diagnostic tests are routinely performed on humans toevaluate the amount or existence of substances present in blood or otherbody fluids. These diagnostic tests typically rely on physiologicalfluid samples removed from a subject, either using a syringe or bypricking the skin. One particular diagnostic test entailsself-monitoring of blood glucose levels by diabetics.

Diabetes is a major health concern, and treatment of the more severeform of the condition, Type I (insulin-dependent) diabetes, requires oneor more insulin injections per day. Insulin controls utilization ofglucose or sugar in the blood and prevents hyperglycemia which, if leftuncorrected, can lead to ketosis. On the other hand, improperadministration of insulin therapy can result in hypoglycemic episodes,which can cause coma and death. Hyperglycemia in diabetics has beencorrelated with several long-term effects of diabetes, such as heartdisease, atherosclerosis, blindness, stroke, hypertension and kidneyfailure.

The value of frequent monitoring of blood glucose as a means to avoid orat least minimize the complications of Type I diabetes is wellestablished. Patients with Type II (non-insulin-dependent) diabetes canalso benefit from blood glucose monitoring in the control of theircondition by way of diet and exercise.

Conventional blood glucose monitoring methods generally require thedrawing of a blood sample (e.g., by fingerprick) for each test, and adetermination of the glucose level using an instrument that readsglucose concentrations by electrochemical or calorimetric methods. TypeI diabetics must obtain several fingerprick blood glucose measurementseach day in order to maintain tight glycemic control. However, the painand inconvenience associated with this blood sampling, along with thefear of hypoglycemia, has led to poor patient compliance, despite strongevidence that tight control dramatically reduces long-term diabeticcomplications. In fact, these considerations can often lead to anabatement of the monitoring process by the diabetic. See, e.g., TheDiabetes Control and Complications Trial Research Group (1993) New Engl.J. Med. 329:977–1036.

Recently, various methods for determining the concentration of bloodanalytes without drawing blood have been developed. For example, U.S.Pat. No. 5,267,152 to Yang et al. describes a noninvasive technique ofmeasuring blood glucose concentration using near-IR radiationdiffuse-reflection laser spectroscopy. Similar near-IR spectrometricdevices are also described in U.S. Pat. No. 5,086,229 to Rosenthal etal. and U.S. Pat. No. 4,975,581 to Robinson et al.

U.S. Pat. No. 5,139,023 to Stanley describes a transdermal blood glucosemonitoring apparatus that relies on a permeability enhancer (e.g., abile salt) to facilitate transdermal movement of glucose along aconcentration gradient established between interstitial fluid and areceiving medium. U.S. Pat. No. 5,036,861 to Sembrowich describes apassive glucose monitor that collects perspiration through a skin patch,where a cholinergic agent is used to stimulate perspiration secretionfrom the eccrine sweat gland. Similar perspiration collection devicesare described in U.S. Pat. No. 5,076,273 to Schoendorfer and U.S. Pat.No. 5,140,985 to Schroeder.

In addition, U.S. Pat. No. 5,279,543 to Glikfeld describes the use ofiontophoresis to noninvasively sample a substance through skin into areceptacle on the skin surface. Glikfeid teaches that this samplingprocedure can be coupled with a glucose-specific biosensor orglucose-specific electrodes in order to monitor blood glucose. Finally,International Publication No. WO 96/00110 to Tamada describes aniontophoretic apparatus for transdermal monitoring of a targetsubstance, wherein an iontophoretic electrode is used to move an analyteinto a collection reservoir and a biosensor is used to detect the targetanalyte present in the reservoir.

SUMMARY OF THE INVENTION

The present invention provides methods and sampling systems formeasuring the concentration of an analyte present in a biologicalsystem. The methods of the invention generally entail sampling anddetecting an analyte from the biological system and deriving adetectable signal therefrom, wherein the signal is specifically relatedto the analyte. The signal can be correlated with a measurement valueindicative of the concentration of analyte present in the biologicalsystem. Sampling system configurations and/or measurement techniques areused to minimize the effect of interfering species on a particularsensing means.

Analyte sampling is carried out using a transdermal sampling system thatis placed in operative contact with a skin or mucosal surface. Inpreferred embodiments, the sampling system transdermally extracts theanalyte from the biological system using iontophoresis. The transdermalsampling system can be maintained in operative contact with the skin ormucosal surface to provide for continual or continuous analytemeasurement.

The analyte can be any specific substance or component that one isdesirous of detecting and/or measuring in a chemical, physical,enzymatic, or optical analysis. Such analytes include, but are notlimited to, amino acids, enzyme substrates or products indicating adisease state or condition, other markers of disease states orconditions, drugs of abuse, therapeutic and/or pharmacologic agents,electrolytes, physiological analytes of interest (e.g., calcium,potassium, sodium, chloride, bicarbonate (CO₂), glucose, urea (bloodurea nitrogen), lactate, hematocrit, and hemoglobin), lipids, and thelike. In preferred embodiments, the analyte is a physiological analyteof interest, for example glucose, or a chemical that has a physiologicalaction, for example a drug or pharmacological agent.

Accordingly, it is an object of the invention to provide a method formeasuring an analyte present in a biological system. The method entailsa step for transdermally extracting the analyte from the biologicalsystem in an extraction step using a sampling system that is inoperative contact with a skin or mucosal surface of the biologicalsystem; and a step for contacting the extracted analyte with a sensormeans in a sensing step to obtain a detectable signal which isspecifically related to the analyte. The extraction and sensing stepsare conducted in a measurement cycle which selectively favorsanalyte-specific signal components over signal components due tointerfering species.

In one aspect of the invention, a method is provided for measuring theconcentration of an analyte present in a biological system. The methodincludes a measurement cycle which comprises an extraction step in whicha sample containing the analyte is transdermally extracted from thebiological system using a sampling system that is in operative contactwith a skin or mucosal surface of the biological system. The method alsoentails a sensing step in which the extracted sample is contacted withsensor means to obtain a measurement signal that is related to analyteconcentration. The measurement cycle further comprises a process forselectively favoring analyte-specific signal components over signalcomponents due to interfering species. Such processes can include (a) adifferential signal process which subtracts non-analyte signalcomponents from the measurement signal, (b) a delay step which isperformed between the extraction and sensing steps, (c) a selectiveelectrochemical detection process which is performed during the sensingstep, (d) a purge step which is performed after the sensing step, (e) acharge segregation step (as in Example 1), or any combination of theprocesses of (a)–(e).

In another aspect of the invention, a method is provided for measuringthe concentration of an analyte present in a biological system. Themethod includes a measurement cycle which comprises transdermallyextracting the analyte from the biological system in an extraction stepusing an iontophoretic sampling system that is in operative contact witha skin or mucosal surface of the biological system, and contacting theextracted analyte with a sensor means in a sensing step to obtain adetectable signal which is specifically related to the analyte. Inparticular, the sampling system comprises (a) a first collectionreservoir containing an ionically conductive medium, a firstiontophoretic sampling means for extracting substances including theanalyte from the biological system into the first collection reservoirto obtain a concentration of the analyte, and a first sensor element,wherein the first sampling means and the first sensor element are inoperative contact with the first collection reservoir; and (b) a secondcollection reservoir containing an ionically conductive medium, a secondiontophoretic sampling means for extracting substances including theanalyte from the biological system into the second collection reservoir,and a second sensor element, wherein the second sampling means and thesecond sensor element are in operative contact with the secondcollection reservoir. The measurement cycle comprises (a) operating thefirst iontophoretic sampling means as an iontophoretic cathode during afirst phase of the extraction step, (b) detecting substances extractedinto the first reservoir with the first sensor element during a firstphase of the sensing step to obtain a first signal, (c) purging residualsignal from the sampling system in a purging step, (d) operating thesecond iontophoretic sampling means as an iontophoretic cathode during asecond phase of the extraction step, and (e) detecting substancesextracted into the second reservoir with the second sensor elementduring a second phase of the sensing step to obtain a second signal. Atleast one of the first and second signals comprises an analyte-specificsignal component.

In one particular embodiment, one of the collection reservoirs includesan enzyme that reacts specifically with the extracted analyte, and thesecond reservoir does not contain the enzyme.

In yet another aspect of the invention, a method is provided formeasuring the concentration of an analyte present in a biologicalsystem. The method includes transdermally extracting the analyte fromthe biological system in an extraction step using an iontophoreticsampling system that comprises first and second collection reservoirswhich are respectively in is operative contact with first and secondiontophoretic sampling means and a skin or mucosal surface of thebiological system. The first and second iontophoretic sampling meansextract substances including the analyte from the biological system intothe first and second collection reservoirs, and the first iontophoreticsampling means is operated as a cathode during the extraction step. Themethod further entails passively collecting substances which diffusefrom, or are secreted by the biological system into a third collectionreservoir using a passive transdermal sampling system that is inoperative contact with a skin or mucosal surface of the biologicalsystem. The analyte that is extracted into the first collectionreservoir is then contacted with a sensing means in a sensing step toobtain an active signal, and the substances collected into the thirdcollection reservoir are contacted with the sensing means to obtain ablank signal. The blank signal is then subtracted from the active signalto provide an analyte-specific signal.

In one aspect of the methods and devices of the present invention, acharge segregation step can be used to reduce the presence ofinterfering species at the sensing means. For example, the measurementcycle can include a charge segregation step wherein sensing takes placeat the cathode and certain interfering species preferentially collect atthe anode, or visa versa.

In a related aspect of the invention, a method is provided for measuringthe concentration of an analyte present in a biological system. Themethod includes transdermally extracting the analyte from the biologicalsystem in an extraction step using an iontophoretic sampling system thatcomprises first, second, and third collection reservoirs which arerespectively in operative contact with first, second, and thirdiontophoretic sampling means and a skin or mucosal surface of thebiological system. The first, second, and third iontophoretic samplingmeans respectively extract substances including the analyte from thebiological system into the first, second, and third collectionreservoirs, and the first and second iontophoretic sampling means areoperated as cathodes during the extraction step. The method furtherentails contacting the analyte extracted into the first collectionreservoir with a sensing means in a sensing step to obtain an activesignal, and contacting the substances extracted into the secondcollection reservoir with the sensing means to obtain a blank signal. Ananalyte-specific signal is then obtained by subtracting the blank signalfrom the active signal.

In a still further related aspect of the invention, a method is providedfor measuring the concentration of an analyte present in a biologicalsystem. The method includes transdermally extracting the analyte fromthe biological system in an extraction step using an iontophoreticsampling system that comprises first, second, and third collectionreservoirs which are respectively in operative contact with first,second, and third iontophoretic sampling means and a skin or mucosalsurface of the biological system. The first, second, and thirdiontophoretic sampling means respectively extract substances includingthe analyte from the biological system into the first, second, and thirdcollection reservoirs, and the first and second iontophoretic samplingmeans are operated as cathodes during the extraction step. The methodfurther entails contacting the analyte extracted into the first andsecond collection reservoirs with a sensing means in a sensing step toobtain multiple analyte-specific signals.

It is another object of the invention to provide a sampling system formeasuring an analyte present in a biological system. The sampling systemcomprises, in operative combination (a) a sampling means for extractingthe analyte from the biological system, (b) a sensing means in operativecontact with the analyte extracted by the sampling means, and (c) amicroprocessor means in operative communication with the sampling andsensing means. The sampling means is adapted for extracting the analyteacross a skin or mucosal surface of a biological system. In preferredembodiments, the sampling means is used to continually or continuouslyextract the analyte. The sensing means is used to obtain a detectablesignal from the extracted analyte, wherein the signal is specificallyrelated to the analyte. The microprocessor means is used to control thesampling means and the sensing means to provide for one or moremeasurement cycles.

In one aspect of the invention, the sampling system comprises (a) afirst collection reservoir containing an ionically conductive medium, afirst iontophoretic sampling means for extracting substances includingthe analyte from the biological system into the first collectionreservoir, and a first sensor element, wherein the first sampling meansand the first sensor element are in operative contact with the firstcollection reservoir; and (b) a second collection reservoir containingan ionically conductive medium, a second iontophoretic sampling meansfor extracting substances including the analyte from the biologicalsystem into the second collection reservoir, and a second sensorelement, wherein the second sampling means and the second sensor elementare in operative contact with the second collection reservoir. In thesubject sampling system, the microprocessor controls a measurement cyclewhich entails (a) operating the first iontophoretic sampling means as aniontophoretic cathode during a first phase of the extraction step, (b)detecting substances extracted into the first reservoir with the firstsensor element during a first phase of the sensing step to obtain afirst signal, (c) purging residual signal from the sampling system in apurging step, (d) operating the second iontophoretic sampling means asan iontophoretic cathode during a second phase of the extraction step,and (e) detecting substances extracted into the second reservoir withthe second sensor element during a second phase of the sensing step toobtain a second signal. At least one of the first and second signalscomprises an analyte-specific signal component.

In a further aspect, the methods and devices of the present inventioncan include enhancement of skin permeability by pricking the skin withmicro-needles when the biological system includes skin, or, for example,a mucosal surface. Such pricking with micro-needles can facilitateextraction an analyte of interest from the biological system.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing disclosure, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a top plan view of an iontophoretic collection reservoirand electrode assembly for use in a transdermal sampling deviceconstructed according to the present invention.

FIG. 1B depicts the side view of the iontophoretic collection reservoirand electrode assembly shown in FIG. 1A.

FIG. 2 is a pictorial representation of an iontophoretic sampling devicewhich includes the iontophoretic collection reservoir and electrodeassembly of FIGS. 1A and 1B.

FIG. 3 is an exploded pictorial representation of components from apreferred embodiment of the automatic sampling system of the presentinvention.

FIG. 4 is a representation of one embodiment of a bimodal electrodedesign. The figure presents an overhead and schematic view of theelectrode assembly 43. In the figure, the bimodal electrode is shown at40 and can be, for example, a Ag/AgCl iontophoretic/counter electrode.The sensing or working electrode (made from, for example, platinum) isshown at 41. The reference electrode is shown at 42 and can be, forexample, a Ag/AgCl electrode. The components are mounted on a suitablenonconductive substrate 44, for example, plastic or ceramic. Theconductive leads 47 leading to the connection pad 45 are covered by asecond nonconductive piece 46 of similar or different material. In thisexample of such an electrode the working electrode area is approximately1.35 cm². The dashed line in FIG. 4 represents the plane of thecross-sectional schematic view presented in FIG. 5.

FIG. 5 is a representation of a cross-sectional schematic view of thebimodal electrodes as they may be used in conjunction with a referenceelectrode and a hydrogel pad. In the figure, the components are asfollows: bimodal electrodes 50 and 51; sensing electrodes 52 and 53;reference electrodes 54 and 55; a substrate 56; and hydrogel pads 57 and58.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular compositionsor biological systems as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “aninterfering species” includes two or more such species, reference to “ananalyte” includes mixtures of analytes, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

Definitions

The terms “analyte” and “target analyte” are used herein to denote anyphysiological analyte of interest that is a specific substance orcomponent that is being detected and/or measured in a chemical,physical, enzymatic, or optical analysis. A detectable signal (e.g., achemical signal or electrochemical signal) can be obtained, eitherdirectly or indirectly, from such an analyte or derivatives thereof.Furthermore, the terms “analyte” and “substance” are usedinterchangeably herein, and are intended to have the same meaning, andthus encompass any substance of interest. In preferred embodiments, theanalyte is a physiological analyte of interest, for example, glucose, ora chemical that has a physiological action, for example, a drug orpharmacological agent.

A “sampling device” or “sampling system” refers to any device forobtaining a sample from a biological system for the purpose ofdetermining the concentration of an analyte of interest. As used herein,the term “sampling” means invasive, minimally invasive or non-invasiveextraction of a substance from the biological system, generally across amembrane such as skin or mucosa. The membrane can be natural orartificial, and can be of plant or animal nature, such as natural orartificial skin, blood vessel tissue, intestinal tissue, and the like.Typically, the sampling means are in operative contact with a“reservoir,” or “collection reservoir,” wherein the sampling means isused for extracting the analyte from the biological system into thereservoir to obtain the analyte in the reservoir. A “biological system”includes both living and artificially maintained systems. Examples ofminimally invasive and noninvasive sampling techniques includeiontophoresis, sonophoresis, suction, electroporation, thermal poration,passive diffusion, microfine (miniature) lances or cannulas,subcutaneous implants or insertions, and laser devices. Sonophoresisuses ultrasound to increase the permeability of the skin (see, e.g.,Menon et al. (1994) Skin Pharmacology 7:130–139). Suitable sonophoresissampling systems are described in International Publication No. WO91/12772, published 5 Sep. 1991. Passive diffusion sampling devices aredescribed, for example, in International Publication Nos.: WO 97/38126(published 16 Oct. 1997); WO 97/42888, WO 97/42886, WO 97/42885, and WO97/42882 (all published 20 Nov. 1997); and WO 97/43962 (published 27Nov. 1997). Laser devices use a small laser beam to burn a hole throughthe upper layer of the patient's skin (see, e.g., Jacques et al. (1978)J. Invest. Dermatology 88:88–93). Examples of invasive samplingtechniques include traditional needle and syringe or vacuum sample tubedevices.

The term “collection reservoir” is used to describe any suitablecontainment means for containing a sample extracted from a biologicalsystem. For example, the collection reservoir can be a receptaclecontaining a material which is ionically conductive (e.g., water withions therein), or alternatively, it can be a material, such as, asponge-like material or hydrophilic polymer, used to keep the water inplace. Such collection reservoirs can be in the form of a hydrogel (forexample, in the form of a disk or pad). Hydrogels are typically referredto as “collection inserts.” Other suitable collection reservoirsinclude, but are not limited to, tubes, vials, capillary collectiondevices, cannulas, and miniaturized etched, ablated or molded flowpaths.

A “housing” for the sampling system can further include suitableelectronics (e.g., microprocessor, memory, display and other circuitcomponents) and power sources for operating the sampling system in anautomatic fashion.

A “monitoring system,” as used herein, refers to a system useful forcontinually or continuously measuring a physiological analyte present ina biological system. Such a system typically includes, but is notlimited to, sampling means, sensing means, and a microprocessor means inoperative communication with the sampling means and the sensing means.

The term “artificial,” as used herein, refers to an aggregation of cellsof monolayer thickness or greater which are grown or cultured in vivo orin vitro, and which function as a tissue of an organism but are notactually derived, or excised, from a pre-existing source or host.

The term “subject” encompasses any warm-blooded animal, particularlyincluding a member of the class Mammalia such as, without limitation,humans and nonhuman primates such as chimpanzees and other apes andmonkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

As used herein, the term “continual measurement” intends a series of twoor more measurements obtained from a particular biological system, whichmeasurements are obtained using a single device maintained in operativecontact with the biological system over the time period in which theseries of measurements is obtained. The term thus includes continuousmeasurements.

The term “transdermal,” as used herein, includes both transdermal andtransmucosal techniques, i.e., extraction of a target analyte acrossskin or mucosal tissue. Aspects of the invention which are describedherein in the context of “transdermal,” unless otherwise specified, aremeant to apply to both transdermal and transmucosal techniques.

The term “transdermal extraction,” or “transdermally extracted” intendsany noninvasive, or at least minimally invasive sampling method, whichentails extracting and/or transporting an analyte from beneath a tissuesurface across skin or mucosal tissue. The term thus includes extractionof an analyte using iontophoresis (reverse iontophoresis),electroosmosis, sonophoresis, microdialysis, suction, and passivediffusion. These methods can, of course, be coupled with application ofskin penetration enhancers or skin permeability enhancing technique suchas tape stripping or pricking with micro-needles. The term“transdermally extracted” also encompasses extraction techniques whichemploy thermal poration, electroporation, microfine lances, microfinecanulas, subcutaneous implants or insertions, and the like.

The term “iontophoresis” intends a method for transporting substancesacross tissue by way of an application of electrical energy to thetissue. In conventional iontophoresis, a reservoir is provided at thetissue surface to serve as a container of material to be transported.Iontophoresis can be carried out using standard methods known to thoseof skill in the art, for example, by establishing an electricalpotential using a direct current (DC) between fixed anode and cathode“iontophoretic electrodes,” alternating a direct current between anodeand cathode iontophoretic electrodes, or using a more complex waveformsuch as applying a current with alternating polarity (AP) betweeniontophoretic electrodes (so that each electrode is alternately an anodeor a cathode).

The term “reverse iontophoresis” refers to the movement of a substancefrom a biological fluid across a membrane by way of an applied electricpotential or current. In reverse iontophoresis, a reservoir is providedat the tissue surface to receive the extracted material.

“Electroosmosis” refers to the movement of a substance through amembrane by way of an electric field-induced convective flow. The termsiontophoresis, reverse iontophoresis, and electroosmosis, will be usedinterchangeably herein to refer to movement of any ionically charged oruncharged substance across a membrane (e.g., an epithelial membrane)upon application of an electric potential to the membrane through anionically conductive medium.

The term “sensing device,” “sensing means,” or “biosensor device”encompasses any device that can be used to measure the concentration ofan analyte, or derivative thereof, of interest. Preferred sensingdevices for detecting blood analytes generally include electrochemicaldevices and chemical devices. Examples of electrochemical devicesinclude the Clark electrode system (see, e.g., Updike, et al., (1967)Nature 214:986–988), and other amperometric, coulometric, orpotentiometric electrochemical devices. Examples of chemical devicesinclude conventional enzyme-based reactions as used in the Lifescan®glucose monitor (Johnson and Johnson, New Brunswick, N.J.) (see, e.g.,U.S. Pat. No. 4,935,346 to Phillips, et al.).

A “biosensor” or “biosensor device” includes, but is not limited to, a“sensor element” which includes, but is not limited to, a “biosensorelectrode” or “sensing electrode” or “working electrode” which refers tothe electrode that is monitored to determine the amount of electricalsignal at a point in time or over a given time period, which signal isthen correlated with the concentration of a chemical compound. Thesensing electrode comprises a reactive surface which converts theanalyte, or a derivative thereof, to electrical signal. The reactivesurface can be comprised of any electrically conductive material suchas, but not limited to, platinum-group metals (including, platinum,palladium, rhodium, ruthenium, osmium, and iridium), nickel, copper,silver, and carbon, as well as, oxides, dioxides, combinations or alloysthereof. Some catalytic materials, membranes, and fabricationtechnologies suitable for the construction of amperometric biosensorswere described by Newman, J. D., et al. (Analytical Chemistry 67(24),4594–4599, 1995).

The “sensor element” can include components in addition to a biosensorelectrode, for example, it can include a “reference electrode,” and a“counter electrode.” The term “reference electrode” is used herein tomean an electrode that provides a reference potential, e.g., a potentialcan be established between a reference electrode and a workingelectrode. The term “counter electrode” is used herein to mean anelectrode in an electrochemical circuit which acts as a current sourceor sink to complete the electrochemical circuit. Although it is notessential that a counter electrode be employed where a referenceelectrode is included in the circuit and the electrode is capable ofperforming the function of a counter electrode, it is preferred to haveseparate counter and reference electrodes because the referencepotential provided by the reference electrode is most stable when it isat equilibrium. If the reference electrode is required to act further asa counter electrode, the current flowing through the reference electrodemay disturb this equilibrium. Consequently, separate electrodesfunctioning as counter and reference electrodes are most preferred.

In one embodiment, the “counter electrode” of the “sensor element”comprises a “bimodal electrode.” The term “bimodal electrode” as usedherein typically refers to an electrode which is capable of functioningnon-simultaneously as, for example, both the counter electrode (of the“sensor element”) and the iontophoretic electrode (of the “samplingmeans”).

The terms “reactive surface,” and “reactive face” are usedinterchangeably herein to mean the surface of the sensing electrodethat: (1) is in contact with the surface of an electrolyte containingmaterial (e.g. gel) which contains an analyte or through which ananalyte, or a derivative thereof, flows from a source thereof; (2) iscomprised of a catalytic material (e.g., carbon, platinum, palladium,rhodium, ruthenium, or nickel and/or oxides, dioxides and combinationsor alloys thereof) or a material that provides sites for electrochemicalreaction; (3) converts a chemical signal (e.g. hydrogen peroxide) intoan electrical signal (e.g., an electrical current); and (4) defines theelectrode surface area that, when composed of a reactive material, issufficient to drive the electrochemical reaction at a-rate sufficient togenerate a detectable, reproducibly measurable, electrical signal thatis correlatable with the amount of analyte present in the electrolyte.

The term “collection reservoir” and “collection insert” are used todescribe any suitable containment means for containing a sampleextracted from a biological system. The reservoir can include a materialwhich is ionically conductive (e.g., water with ions therein), whereinanother material such as a sponge-like material or hydrophilic polymeris used to keep the water in place. Such collection reservoirs can be inthe form of a hydrogel (for example, in the shape of a disk or pad).Other suitable collection reservoirs include, but are not limited to,tubes, vials, capillary collection devices, cannulas, and miniaturizedetched, ablated or molded flow paths.

An “ionically conductive material” refers to any material that providesionic conductivity, and through which electrochemically active speciescan diffuse. The ionically conductive material can be, for example, asolid, liquid, or semi-solid (e.g., in the form of a gel) material thatcontains an electrolyte, which can be composed primarily of water andions (e.g., sodium chloride), and generally comprises 50% or more waterby weight. The material can be in the form of a gel, a sponge or pad(e.g., soaked with an electrolytic solution), or any other material thatcan contain an electrolyte and allow passage therethrough ofelectrochemically active species, especially the analyte of interest.

The term “physiological effect” encompasses effects produced in thesubject that achieve the intended purpose of a therapy. In preferredembodiments, a physiological effect means that the symptoms of thesubject being treated are prevented or alleviated. For example, aphysiological effect would be one that results in the prolongation ofsurvival in a patient.

A “laminate” as used herein, refers to structures comprised of at leasttwo bonded layers. The layers may be bonded by welding or through theuse of adhesives. Examples of welding include, but are not limited to,the following: ultrasonic welding, heat bonding, and inductively coupledlocalized heating followed by localized flow. Examples of commonadhesives include, but are not limited to, pressure sensitive adhesives,thermoset adhesives, cyanocrylate adhesives, epoxies, contact adhesives,and heat sensitive adhesives.

A “collection assembly”, as used herein, refers to structures comprisedof several layers, where the assembly includes at least one collectioninsert, for example a hydrogel. An example of a collection assembly ofthe present invention is a mask layer, collection inserts, and aretaining layer where the layers are held in appropriate, functionalrelationship to each other but are not necessarily a laminate, i.e., thelayers may not be bonded together. The layers may, for example, be heldtogether by interlocking geometry or friction.

An “autosensor assembly” as used herein, refers to structures generallycomprising a mask layer, collection inserts, a retaining layer, anelectrode assembly, and a support tray. The autosensor assembly may alsoinclude liners. The layers of the assembly are held in appropriate,functional relationship to each other.

The mask and retaining layers are preferably composed of materials thatare substantially impermeable to the analyte (chemical signal) to bedetected (e.g., glucose); however, the material can be permeable toother substances. By “substantially impermeable” is meant that thematerial reduces or eliminates chemical signal transport (e.g., bydiffusion). The material can allow for a low level of chemical signaltransport, with the proviso that the chemical signal that passes throughthe material does not cause significant edge effects at the sensingelectrode.

“Substantially planar” as used herein, includes a planar surface thatcontacts a slightly curved surface, for example, a forearm or upper armof a subject. A “substantially planar” surface is, for example, asurface having a shape to which skin can conform, i.e., contactingcontact between the skin and the surface.

By the term “printed” as used herein is meant a substantially uniformdeposition of an electrode formulation onto one surface of a substrate(i.e., the base support). It will be appreciated by those skilled in theart that a variety of techniques may be used to effect substantiallyuniform deposition of a material onto a substrate, e.g., Gravure-typeprinting, extrusion coating, screen coating, spraying, painting, or thelike.

The term “signal-to-noise ratio” describes the relationship between theactual signal intended to be measured and the variation in signal in theabsence of the analyte. The terms “S/N” and “SNR” are also used to referto the signal-to-noise ratio. “Noise,” as used herein, refers to anyundesirable signal which is measured along with the intended signal.

As used herein, an “interfering species” is broadly defined as anymoiety in an extracted sample that gives rise to signal noise and whichis not a target analyte. The term thus includes any component of anextracted sample that provides a signal not related to the targetanalyte.

The term “enzyme” intends any compound or material which catalyzes areaction between molecules to produce one or more reaction products. Theterm thus includes protein enzymes, or enzymatically active portions(fragments) thereof, which proteins and/or protein fragments may beisolated from a natural source, or recombinantly or syntheticallyproduced. The term also encompasses designed synthetic enzyme mimetics.

General Methods

The present invention relates to the use of a device for transdermallyextracting and measuring the concentration of a target analyte presentin a biological system. In preferred embodiments, the sensing devicecomprises a biosensor. The sampling device is used to extract smallamounts of a target analyte from the biological system, and then senseand/or quantify the concentration of the target analyte. Measurementwith the biosensor and/or sampling with the sampling device can becarried out in a continual or continuous manner. In one particularembodiment, a biosensor is used which comprises an electrochemicalsensing element.

The analyte can be any specific substance or component that one isdesirous of detecting and/or measuring in a chemical, physical,enzymatic, or optical analysis. Such analytes include, but are notlimited to, amino acids, enzyme substrates or products indicating adisease state or condition, other markers of disease states orconditions, drugs of abuse, therapeutic and/or pharmacologic agents(e.g., theophylline, anti-HIV drugs, lithium, anti-epileptic drugs,cyclosporin, chemotherapeutics), electrolytes, physiological analytes ofinterest (e.g., urate/uric acid, carbonate, calcium, potassium, sodium,chloride, bicarbonate (CO₂), glucose, urea (blood urea nitrogen),lactate/lactic acid, hydroxybutyrate, cholesterol, triglycerides,creatine, creatinine, insulin, hematocrit, and hemoglobin), blood gases(carbon dioxide, oxygen, pH), lipids, heavy metals (e.g., lead, copper),and the like. In preferred embodiments, the analyte is a physiologicalanalyte of interest, for example glucose, or a chemical that has aphysiological action, for example a drug or pharmacological agent.

In order to facilitate detection of the analyte, an enzyme can bedisposed in the collection reservoir, or, if several collectionreservoirs are used, the enzyme can be disposed in several or all of thereservoirs. The selected enzyme is capable of catalyzing a reaction withthe extracted analyte (in this case glucose) to the extent that aproduct of this reaction can be sensed, e.g., can be detectedelectrochemically from the generation of a current which current isdetectable and proportional to the concentration or amount of theanalyte which is reacted. A suitable enzyme is glucose oxidase whichoxidizes glucose to gluconic acid and hydrogen peroxide. The subsequentdetection of hydrogen peroxide on an appropriate biosensor electrodegenerates two electrons per hydrogen peroxide molecule which create acurrent which can be detected and related to the amount of glucoseentering the device. Glucose oxidase (GOx) is readily availablecommercially and has well known catalytic characteristics. However,other enzymes can also be used, so long as they specifically catalyze areaction with an analyte or substance of interest to generate adetectable product in proportion to the amount of analyte so reacted.

In like manner, a number of other analyte-specific enzyme systems can beused in the invention, which enzyme systems operate on much the samegeneral techniques. For example, a biosensor electrode that detectshydrogen peroxide can be used to detect ethanol using an alcohol oxidaseenzyme system, or similarly uric acid with urate oxidase system, ureawith a urease system, cholesterol with a cholesterol oxidase system, andtheophylline with a xanthine oxidase system.

In addition, the oxidase enzyme (used for hydrogen peroxide-baseddetection) can be replaced with another redox system, for example, thedehydrogenase-enzyme NAD-NADH, which offers a separate route todetecting additional analytes. Dehydrogenase-based sensors can useworking electrodes made of gold or carbon (via mediated chemistry).Examples of analytes suitable for this type of monitoring include, butare not limited to, cholesterol, ethanol, hydroxybutyrate,phenylalanine, triglycerides, and urea. Further, the enzyme can beeliminated and detection can rely on direct electrochemical orpotentiometric detection of an analyte. Such analytes include, withoutlimitation, heavy metals (e.g., cobalt, iron, lead, nickel, zinc),oxygen, carbonate/carbon dioxide, chloride, fluoride, lithium, pH,potassium, sodium, and urea. Also, the sampling system described hereincan be used for therapeutic drug monitoring, for example, monitoringanti-epileptic drugs (e.g., phenylion), chemotherapy (e.g., adriamycin),hyperactivity (e.g., ritalin), and anti-organ-rejection (e.g.,cyclosporin).

One problem related to the use of transdermal extraction techniquesrelates to the presence of interfering species within an extractedsample, which species may be extracted by the transdermal samplingprocess, diffuse through the skin passively, and/or be secreted inperspiration or sebum. These interfering species (that is, any speciesdetected by a sensor which is not the analyte of interest, or anyspecies which interferes with, or reduces an analyte-specific signal)can give rise to significant background interferences, decreasing theoverall sensor signal-to-noise response. In addition, since transdermalsampling techniques generally extract very small quantities of analyte,sensor devices which are used for sensing and/or quantitating theextracted analyte need to perform reliably and reproducibly using smallanalyte concentrations (e.g., sub-mM) which are well below thosemeasured by conventional detection techniques (which are generally inthe mM range). As used herein, “sub-mM” refers to any concentration thatis less than 1 mM. Thus, background interferences which may not beproblematic in other applications may produce critical interferenceswith transdermal sampling systems which measure sub-mM concentrations ofextracted analyte. The present invention is concerned with the reductionof such interferences.

Accordingly, in one embodiment of the invention, a method is providedfor measuring the concentration of an analyte present in a biologicalsystem. The method includes a measurement cycle having an extractionstep in which a sample containing the analyte is transdermally extractedfrom the biological system using a sampling system that is in operativecontact with a skin or mucosal surface of said biological system. Asensing step is also carried out during the measurement cycle. In thesensing step, the extracted sample is contacted with a sensor to obtaina measurement signal that is related to analyte concentration. Themeasurement cycle further comprises a process for selectively favoringanalyte-specific signal components over signal components due tointerfering species, wherein the process can entail (a) a differentialsignal process which subtracts non-analyte signal components from themeasurement signal, (b) a delay step which is performed between theextraction and sensing steps, (c) a selective electrochemical detectionprocess which is performed during the sensing step, (d) a purge stepwhich is performed after the sensing step, (e) a charge segregation step(e.g., uric acid migrates to the anode, and glucose to the cathode, asin Example 1), and (f) any combination of the processes of (a)–(e).

The transdermal sampling device used to extract the sample can employany suitable minimally invasive or noninvasive sampling techniqueincluding, for example, iontophoresis, sonophoresis, suction,electroporation, thermal poration, passive diffusion, use of microfine(miniature) lances or cannulas, use of subcutaneous implants orinsertions, and use of laser devices. The sensing step can be carriedout using any suitable sensing methodology, for example, colormetric,chemical (e.g., enzyme-based reactions) or electrochemical (e.g.,amperometric, coulometric, or potentiometric) methods.

The differential signal process can be carried out in several ways. Forexample, the extracted sample can be divided into two portions, whereina first portion is contacted with the sensor to obtain a signal thatpredominantly contains signal components due to interfering species andbackground variations. The second portion is contacted with the sensorto obtain a measurement signal that contains analyte-specific signalcomponents, and the first signal is then subtracted from the second inorder to obtain a refined signal that is specifically related to theanalyte. An analyte-specific signal can be readily obtained using aenzyme which reacts specifically with the analyte to produce adetectable signal. For example, if the analyte is glucose, the glucoseoxidase enzyme can be contacted with the second sample portion. Thisenzyme catalyzes a reaction with glucose to generate hydrogen peroxide,which can then be detected using conventional electrochemicalbiosensors.

Alternatively, the differential signal process can be carried out byextracting two samples from the biological system, wherein at least oneof the samples contains the target analyte. The samples are thencontacted with a suitable sensor, whereby a first signal is obtainedfrom one of the samples and contains predominantly signal components dueto interfering species and background variations (e.g., a blank signal).A second signal is obtained from the other sample and containsanalyte-specific signal components (e.g., an active signal). Subtractionof the first signal from the second provides a refined signal that isspecifically related to the analyte. Here again, an analyte-specificsignal can be readily obtained using a enzyme which reacts specificallywith the analyte in the second sample to produce a detectable signal.The first sample can be obtained using any transdermal samplingtechnique, including both passive and active collection techniques,while the second sample is generally obtained using active samplingtechniques, e.g., iontophoresis or sonophoresis. The sensor or sensorswhich are used to obtain the signals can, of course, be attached to, orbe integral with the sampling device used in the extraction step, or canbe part of a discrete sensing apparatus.

The selective electrochemical detection process can be carried out asfollows. Electrochemical detection using biosensor electrodes allows fora variety of selective detection processes, wherein the electrodes canbe operated at lowered or raised potentials in order to favor detectionof analyte-specific electrochemical signal over signal due tointerfering species or background variance. For example, operating anelectrochemical biosensor electrode at a potential of about 0.5V or lessallows one to avoid signal components due to common interfering speciescollected with transdermal sampling devices.

In each of the above methods, the sample or samples can be extractedinto a suitable collection reservoir(s) placed in operative contact withthe biological system (e.g., contacted with a skin or mucosal surface ofa subject). In addition, a sensor can be included with each collectionreservoir to detect extracted substances. In one preferred embodiment,the transdermal sampling device is maintained in contact with thebiological system in order to provide for continual or continuousmonitoring of the analyte concentration.

In the context of such continual or continuous monitoring of the analyte(e.g., where the sampling device is maintained in contact with thesystem to obtain two or more samples and analyte measurements), themethod can further include a purge step wherein residual signal isreduced or substantially eliminated from the sampling system (e.g., fromone or more collection reservoir) between measurements. If required, thepurge step can be carried out using an electrochemical sensor electrodewhich is contacted with the reservoir during the sensing operation.Further, the electrode can be operated at a raised potential in order toensure that residual signal components are removed from the samplingsystem. Exemplary purge techniques are described in detail herein below.An optional delay step can also be included between the extraction andsensing steps in the subject method in order to improve thesignal-to-noise ratio in detected signals.

Although all of the methods of the invention are broadly applicable tosampling any chemical analyte and/or substance, the invention isexpressly exemplified hereafter for use in transdermal sampling andquantifying or qualifying glucose or a glucose metabolite.

Accordingly, in another embodiment of the invention, an automaticsampling system is used to monitor levels of glucose in a biologicalsystem. The method is carried out using a sampling system (device) whichtransdermally extracts glucose from the system, in this case, an animalsubject. Transdermal extraction is carried out by applying an electricalcurrent to a tissue surface at a collection site. The electrical currentis used to extract small amounts of glucose from the subject into one ormore collection reservoirs. At least one collection reservoir is incontact with a biosensor which provides for measurement of glucoseconcentration in the subject.

More particularly, a collection reservoir is contacted with a tissuesurface, for example, the stratum corneum of a patient's skin. Anelectrical force is then applied to the tissue surface in order toextract glucose or a glucose metabolite from the tissue into thecollection reservoir. Extraction is typically performed for a period ofabout 1–20 minutes. If desired, however, extraction can be carried outcontinually or continuously over a period of about 1–24 hours, orlonger. After extraction, the collection reservoir is analyzed tomeasure the amount or concentration of glucose or glucose metabolitecontained therein. The measured value correlates with the subject'sblood glucose level. If continual or continuous sampling is desired, thecollection reservoir is analyzed periodically or continuously to measurethe glucose analyte.

In certain embodiments of the invention, one or more additionalcollection reservoirs are used in the transdermal sampling system. Theseadditional collection reservoirs are also contacted with an electrodewhich generates a current sufficient to extract substances from thetissue into the collection reservoir.

The collection reservoirs which are used with the iontophoretic samplingsystem generally contain an ionically conductive liquid orliquid-containing medium. The conductive medium is preferably a hydrogelwhich can contain ionic substances in an amount sufficient to producehigh ionic conductivity. The hydrogel is formed from a solid or liquidmaterial (solute) which, when combined with water, forms a gel by theformation of a structure which holds water including interconnectedcells and/or network structure formed by the solute. Suitable hydrogelformulations are described in International Publication Nos. WO97/02811, published 30 Jan. 1997, and WO 96/00110, published 4 Jan.1996, each of which publications are incorporated herein by reference intheir entireties.

Since the sampling system of the present invention is intended to beoperated at very low (electrochemical) background noise levels, allcomponents of the system which will be in contact with extracted sample(e.g., the collection reservoirs) preferably do not contain orcontribute significant electrochemically sensitive components and/orcontaminants. Thus, components of the system are generally formulatedusing a judicious selection of materials and reagents which do not addsignificant amounts of electrochemical contaminants to the final system.

In order to facilitate detection of the analyte, an enzyme can bedisposed in the collection reservoir, or, if several collectionreservoirs are used, the enzyme can be disposed in several or all of thereservoirs. The selected enzyme is capable of catalyzing a reaction withthe extracted analyte (in this case glucose) to the extent that aproduct of this reaction can be sensed, e.g., can be detectedelectrochemically from the generation of a current which current isdetectable and proportional to the concentration or amount of theanalyte which is reacted. A suitable enzyme is glucose oxidase whichoxidizes glucose to gluconic acid and hydrogen peroxide. The subsequentdetection of hydrogen peroxide on an appropriate biosensor electrodegenerates two electrons per hydrogen peroxide molecule which create acurrent which can be detected and related to the amount of glucoseentering the device. Glucose oxidase (GOx) is readily availablecommercially and has well known catalytic characteristics. However,other enzymes can also be used, so long as they specifically catalyze areaction with an analyte or substance of interest to generate adetectable product in proportion to the amount of analyte so reacted.

In like manner, a number of other analyte-specific enzyme systems can beused in the invention, which enzyme systems operate on much the samegeneral techniques. For example, a biosensor electrode that detectshydrogen peroxide can be used to detect ethanol using an alcohol oxidaseenzyme system, or similarly uric acid with urate oxidase system,cholesterol with a cholesterol oxidase system, and theophylline with axanthine oxidase system.

Referring again to the glucose sampling system exemplified herein, asthe glucose analyte is transdermally extracted into the collectionreservoir, it reacts with the GOx within the reservoir to producehydrogen peroxide The presence of hydrogen peroxide generates a currentat the biosensor electrode that is directly proportional to the amountof hydrogen peroxide in the reservoir. This current provides a signalwhich can be detected and interpreted by an associated system controllerto provide a glucose concentration value for display.

If desired, the detected current can be correlated with the subject'sblood glucose concentration so that the system controller displays thesubject's actual blood glucose concentration as measured by the samplingsystem. For example, the system can be calibrated to the subject'sactual blood glucose concentration by sampling the subject's bloodduring a standard glucose tolerance test, and analyzing the bloodglucose using both a standard blood glucose monitor and the samplingsystem of the present invention. In this manner, measurements obtainedby the sampling system can be S correlated to actual values using knownstatistical techniques.

Iontophoretic extraction can be carried out as follows. A firstiontophoretic electrode is contacted with the collection reservoir(which is in contact with a target tissue surface), and a secondiontophoretic electrode is contacted with either a second collectionreservoir in contact with the tissue surface, or some other ionicallyconductive medium in contact with the tissue. A power source provides anelectric potential between the two electrodes to perform reverseiontophoresis in a manner known in the art.

An electric potential (either direct current or a more complex waveform)is applied between the two iontophoresis electrodes such that currentflows from the first electrode through the first conductive medium intothe skin, and back out from the skin through the second conductivemedium to the second electrode. This current flow extracts substancesthrough the skin into the one or more collection reservoirs through theprocess of reverse iontophoresis or electroosmosis. The electricpotential may be applied as described in International Publication No.WO 96/00110, published 4 Jan. 1996. Any suitable iontophoretic electrodesystem can be employed, however it is preferred that a silver/silverchloride (Ag/AgCl) electrode system is used.

Referring now to FIGS. 1A and 1B, one particular iontophoreticcollection reservoir and electrode assembly for use in such transdermalsensing devices is generally indicated at 2. The assembly comprises twoiontophoretic collection reservoirs, 4 and 6, each having a conductivemedium 8, and 10 (preferably cylindrical hydrogel pads), respectivelydisposed therein. First (12) and second (14) ring-shaped iontophoreticelectrodes are respectively contacted with conductive medium 8 and 10.The first iontophoretic electrode 12 surrounds three biosensorelectrodes which are also contacted with the conductive medium 8, aworking electrode 16, a reference electrode 18, and a counter electrode20. A guard ring 22 separates the biosensor electrodes from theiontophoretic electrode 12 to minimize noise from the iontophoreticcircuit. Conductive contacts provide communication between theelectrodes and an associated power source and control means as describedin detail below. A similar biosensor electrode arrangement can becontacted with the conductive medium 10, or the medium can not have asensor means contacted therewith.

Referring now to FIG. 2, the iontophoretic collection reservoir andelectrode assembly 2 of FIGS. 1A and 1B is shown in exploded view incombination with a suitable iontophoretic sampling device housing 32.The housing can be a plastic case or other suitable structure whichpreferably is configured to be worn on a subjects arm in a mannersimilar to a wrist watch. As can be seen, conductive media 8 and 10(hydrogel pads) are separable from the assembly 2; however, when theassembly 2 and the housing 32 are assembled to provide an operationaliontophoretic sampling device 30, the media are in contact with theelectrodes to provide a electrical contact therewith.

Referring now to FIG. 3, an exploded view of the key components from oneembodiment of an iontophoretic sampling system is presented. Thesampling system components include two biosensor/iontophoretic electrodeassemblies, 304 and 306, each of which have an annular iontophoreticelectrode, respectively indicated at 308 and 310, which encircles abiosensor 312 and 314. The electrode assemblies 304 and 306 are printedonto a polymeric substrate 316 which is maintained within a sensor tray318. A collection reservoir assembly 320 is arranged over the electrodeassemblies, wherein the collection reservoir assembly comprises twohydrogel inserts 322 and 324 retained by a gel retaining layer 326 and amask layer 328.

In one embodiment, the electrode assemblies can include bimodalelectrodes as shown in FIG. 4.

The components shown in exploded view in FIG. 3 are intended for use ina automatic sampling device which is configured to be worn like anordinary wristwatch. As described in International Publication No. WO96/00110, published 4 Jan. 1996, the wristwatch housing (not shown)contains conductive leads which communicate with the iontophoreticelectrodes and the biosensor electrodes to control cycling and providepower to the iontophoretic electrodes, and to detect electrochemicalsignals produced at the biosensor electrode surfaces. The wristwatchhousing can further include suitable electronics (e.g., microprocessor,memory, display and other circuit components) and power sources foroperating the automatic sampling system.

Modifications and additions to the embodiment of FIG. 3 will be apparentto those skilled in the art in light of the teachings of the presentspecification.

A power source (e.g., one or more rechargeable or nonrechargeablebatteries) can be disposed within the housing 32 or within the straps 34which hold the device in contact with a skin or mucosal surface of asubject.

In use, an electric potential (either direct current or a more complexwaveform) is applied between the two is iontophoretic electrodes 12 and14 such that current flows from the first iontophoretic electrode 12,through the first conductive medium 8 into the skin or mucosal surface,and then back out through the second conductive medium 10 to the secondiontophoretic electrode 14. The current flow is sufficient to extractsubstances including an analyte of interest through the skin into one orboth of collection reservoirs 4 and 6. The electric potential may beapplied using any suitable technique, for example, the applied currentdensity may be in the range of about 0.01 to 0.5 mA/cm². In a preferredembodiment, the device is used for continual or continuous monitoring,and the polarity of iontophoretic electrodes 12 and 14 is alternated ata rate of about one switch every 10 seconds to about one switch everyhour so that each electrode is alternately a cathode or an anode. Thehousing 32 can further include an optional temperature sensing element(e.g., a thermistor, thermometer, or thermocouple device) which monitorsthe temperature at the collection reservoirs to enable temperaturecorrection of sensor signals. The housing can further include anoptional conductance sensing element (e.g., an integrated pair ofelectrodes) which monitors conductance at the skin or mucosal surface toenable data screening correction or invalidation of sensor signals.

After a suitable iontophoretic extraction period, one or both of thesensor electrode sets can be activated in order to detect extractedsubstances including the analyte of interest.

Further, the sampling device can operate in an alternating polarity modeusing first and second bimodal electrodes (FIGS. 5, 50 and 51) and twocollection reservoirs (FIGS. 5, 57 and 58). Each bi-modal electrode(FIGS. 4, 40; FIGS. 5, 50 and 51) serves two functions depending on thephase of the operation: (1) an electro-osmotic electrode (oriontophoretic electrode) used to electrically draw analyte from a sourceinto a collection reservoir comprising water and an electrolyte, and tothe area of the electrode subassembly; and (2) as a counter electrode tothe first sensing electrode at which the chemical compound iscatalytically converted at the face of the sensing electrode to producean electrical signal.

The reference (FIGS. 5, 54 and 55; FIGS. 4, 42) and sensing electrodes(FIGS. 5, 52 and 53; FIGS. 4, 41), as well as, the bimodal electrode(FIGS. 5, 50 and 51; FIGS. 4, 40) are connected to a standardpotentiostat circuit during sensing. In general, practical limitationsof the system require that the bimodal electrode will not act as both acounter and iontophoretic electrode simultaneously.

The general operation of an iontophoretic sampling system in thisembodiment is the cyclical repetition of two phases: (1) areverse-iontophoretic phase, followed by a (2) sensing phase. During thereverse iontophoretic phase, the first bimodal electrode (FIGS. 5, 50)acts as an iontophoretic cathode and the second bimodal electrode (FIGS.5, 51) acts as an iontophoretic anode to complete the circuit. Analyteis collected in the reservoirs, for example, a hydrogel (FIGS. 5, 57 and58). At the end of the reverse iontophoretic phase, the iontophoreticcurrent is turned off. During the sensing phase, in the case of glucose,a potential is applied between the reference electrode (FIGS. 5, 54) andthe sensing electrode (FIGS. 5, 52). The chemical signal reactscatalytically on the catalytic face of the first sensing electrode(FIGS. 5, 52) producing an electrical current, while the first bi-modalelectrode (FIGS. 5, 50) acts as a counter electrode to complete theelectrical circuit.

The electrode described is particularly adapted for use in conjunctionwith a hydrogel collection reservoir system for monitoring glucoselevels in a subject through the reaction of collected glucose with theenzyme glucose oxidase present in the hydrogel matrix.

The bi-modal electrode is preferably comprised of Ag/AgCl. Theelectrochemical reaction which occurs at the surface of this electrodeserves as a facile source or sink for electrical current. This propertyis especially important for the iontophoresis function of the electrode.Lacking this reaction, the iontophoresis current could cause thehydrolysis of water to occur at the iontophoresis electrodes causing pHchanges and possible gas bubble formation. The pH changes to acidic orbasic pH could cause skin irritation or burns. The ability of an Ag/AgClelectrode to easily act as a source of sink current is also an advantagefor its counter electrode function. For a three electrodeelectrochemical cell to function properly, the current generationcapacity of the counter electrode should not limit the speed of thereaction at the sensing electrode. In the case of a large sensingelectrode, the counter electrode should be able to sourceproportionately larger currents.

The design of the sampling system provides for a larger sensingelectrode (see for example, FIG. 4) than previously designed.Consequently, the size of the bimodal electrode should be sufficient sothat when acting as a counter electrode with respect to the sensingelectrode the counter electrode does not become limiting the rate ofcatalytic reaction at the sensing electrode catalytic surface.

Two methods exist to ensure that the counter electrode does not limitthe current at the sensing electrode: (1) the bi-modal electrode is mademuch larger than the sensing electrode, or (2) a facile counter reactionis provided.

During the reverse iontophoretic phase, the power source provides acurrent flow to the first bi-modal electrode to facilitate theextraction of the chemical signal into the reservoir. During the sensingphase, the power source is used to provide voltage to the first sensingelectrode to drive the conversion of chemical signal retained inreservoir to electrical signal at the catalytic face of the sensingelectrode. The power source also maintains a fixed potential at theelectrode where, for example hydrogen peroxide is converted to molecularoxygen, hydrogen ions, and electrons, which is compared with thepotential of the reference electrode during the sensing phase. While onesensing electrode is operating in the sensing mode it is electricallyconnected to the adjacent bimodal electrode which acts as a counterelectrode at which electrons generated at the sensing electrode areconsumed.

The electrode sub-assembly can be operated by electrically connectingthe bimodal electrodes such that each electrode is capable offunctioning as both an iontophoretic electrode and counter electrodealong with appropriate sensing electrode(s) and reference electrode(s),to create standard potentiostat circuitry.

A potentiostat is an electrical circuit used in electrochemicalmeasurements in three electrode electrochemical cells. A potential isapplied between the reference electrode and the sensing electrode. Thecurrent generated at the sensing electrode flows through circuitry tothe counter electrode (i.e., no current flows through the referenceelectrode to alter its equilibrium potential). Two independentpotentiostat circuits can be used to operate the two biosensors. For thepurpose of the present sampling system, the electrical current measuredat the sensing electrode subassembly is the current that is correlatedwith an amount of chemical signal.

With regard to continual operation for extended periods of time, Ag/AgClelectrodes are provided herein which are capable of repeatedly forming areversible couple which operates without unwanted electrochemical sidereactions (which could give rise to changes in pH, and liberation ofhydrogen and oxygen due to water hydrolysis). The Ag/AgCl electrodes ofthe present sampling system are thus formulated to withstand repeatedcycles of current passage in the range of about 0.01 to 1.0 mA per cm²of electrode area. With regard to high electrochemical purity, theAg/AgCl components are dispersed within a suitable polymer binder toprovide an electrode composition which is not susceptible toattack-(e.g., plasticization) by components in the collection reservoir,e.g., the hydrogel composition. The electrode compositions are alsoformulated using analytical- or electronic-grade reagents and solvents,and the polymer binder composition is selected to be free ofelectrochemically active contaminants which could diffuse to thebiosensor to produce a background current.

Because the Ag/AgCl iontophoretic electrodes must be capable ofcontinual cycling over extended periods of time, the absolute amounts ofAg and AgCl available in the electrodes, and the overall Ag/AgClavailability ratio, can be adjusted to provide for the passage of highamounts of charge. Although not limiting in the sampling systemdescribed herein, the Ag/AgCl ratio can approach unity. In order tooperate within the preferred system which uses a biosensor having ageometric area of 0.1 to 3 cm², the iontophoretic electrodes areconfigured to provide an approximate electrode area of 0.3 to 1.0 cm²,preferably about 0.85 cm². These electrodes provide for reproducible,repeated cycles of charge passage at current densities ranging fromabout 0.01 to 1.0 mA/cm² of electrode area. More particularly,electrodes constructed according to the above formulation parameters,and having an approximate electrode area of 0.85 cm², are capable of areproducible total charge passage (in both anodic and cathodicdirections) of 270 mC, at a current of about 0.3 mA (current density of0.35 mA/cm²) for at least about 48 cycles in a 24 hour period.

Once formulated, the Ag/AgCl electrode composition-is affixed to asuitable rigid or flexible nonconductive surface as described above withrespect to the biosensor electrode composition. A silver (Ag) underlayeris first applied to the surface in order to provide uniform conduction.The Ag/AgCl electrode composition is then applied over the Ag underlayerin any suitable pattern or geometry using various thin film techniques,such as sputtering, evaporation, vapor phase deposition, or the like, orusing various thick film techniques, such as film laminating,electroplating, or the like. Alternatively, the Ag/AgCl composition canbe applied using screen printing, pad printing, inkjet methods, transferroll printing, or similar techniques. Preferably, both the Ag underlayerand the Ag/AgCl electrode are applied using a low temperature screenprint onto a polymeric substrate. This low temperature screen print canbe carried out at about 125 to 160° C., and the screening can be carriedout using a suitable mesh, ranging from about 100–400 mesh.

In one embodiment of the present invention, the sampling system can havetwo collection reservoirs which contain, for example, an activecollection reservoir, having the GOx enzyme, and a blank collectionreservoir (without the GOx enzyme); or, in an alternative, two activereservoirs, i.e., two reservoirs containing the GOx enzyme. In the caseof an active collection reservoir and a blank collection reservoirsignal can be adjusted by subtraction of the blank reservoir signal fromthe signal obtained from the active reservoir. In the case of two activecollection reservoirs the signals can be summed and averaged, or a totalof the two signals can be used. This signal, for example the detectedcurrent, is then used alone or in combination with other factors (forexample, glucose concentration at a calibration point, skin temperature,conductivity, voltage, time since calibration of the system, etc.) toprovide a glucose concentration value. The measurement cycle can furtherinclude a process for selectively favoring analyte-specific signalcomponents over signal components due to interfering species (asdescribed below). Such process can include, but are not limited to, (a)a differential signal process which subtracts non-analyte signalcomponents from the measurement signal, (b) a delay step which isperformed between the extraction and sensing steps, (c) a selectiveelectrochemical detection process which is performed during the sensingstep, (d) a purge step which is performed after the sensing step, (e) acharge segregation step (e.g., uric acid migrates to the anode, andglucose to the cathode, as in Example 1), and (f) any combinations of(a) to (e).

In particular embodiments, the detected current can be correlated withthe subject's blood glucose concentration (typically using statisticalalgorithms associated with a microprocessor) so that the systemcontroller may display the subject's actual blood glucose concentrationas measured by the sampling system. For example, the system can becalibrated to the subject's actual blood glucose concentration bysampling the subject's blood during a standard glucose tolerance test,and analyzing the blood glucose using both a standard blood glucosemonitor and the sampling system of the present invention. In addition oralternately, the sampling system can be calibrated at a calibration timepoint where the signal obtained from the sampling system at that timepoint is correlated to blood glucose concentration at that time point asdetermined by direct blood testing (for example, glucose concentrationcan be determined using a HemoCue® clinical analyzer (HemoCue AB,Sweden)). In this manner, measurements obtained by the sampling systemcan be correlated to actual values using known statistical techniques.Such statistical techniques can be formulated as algorithms(s) andincorporated in a microprocessor associated with the sampling system.

Operation of the iontophoretic sampling device 30 is optionallycontrolled by a controller 36 (e.g., a microprocessor), which interfaceswith the iontophoretic electrodes, the sensor electrodes, the powersupply, the optional temperature and/or conductance sensing elements, adisplay and other electronics. For example, the controller 36 caninclude a programmable a controlled circuit source/sink drive fordriving the iontophoretic electrodes. Power and reference voltage areprovided to the sensor electrodes, and signal amplifiers can be used toprocess the signal from the working electrode or electrodes. In general,the controller discontinues the iontophoretic current drive duringsensing periods. A sensor confidence loop can be provided forcontinually monitoring the sampling system to ensure proper operations.

User control can be carried out using push buttons located on thehousing 32, and an optional liquid crystalline display (LCD) can providevisual prompts, readouts and visual alarm indications. Themicroprocessor generally uses a series of programmed sequences tocontrol the operations of the sampling device, which sequences can bestored in the microprocessor's read only memory (ROM). Embedded software(firmware) controls activation of measurement and display operations,calibration of analyte readings, setting and display of high and lowanalyte value alarms, display and setting of time and date functions,alarm time, and display of stored readings. Sensor signals obtained fromthe sensor electrodes can be processed before storage and display by oneor more signal processing functions or algorithms provided by theembedded software. The microprocessor can also include an electronicallyerasable, programmable, read only memory (EEPROM) for storingcalibration parameters, user settings and all downloadable sequences.

Further, the sampling system can be pre-programmed to begin execution ofits signal measurements (or other functions) at a designated time. Oneapplication of this feature is to have the sampling system in contactwith a subject and to program the sampling system to begin sequenceexecution during the night so that it is available for calibrationimmediately upon waking. One advantage of this feature is that itremoves any need to wait for the sampling system to warm-up beforecalibrating it.

As discussed above, the sensitivity of such iontophoretic extraction andelectrochemical detection methodologies can be affected by the presenceof interfering species within an extracted sample. These interferingspecies may be extracted by the iontophoretic sampling process, diffusethrough the skin passively, or may be extracted in perspiration orsebum, and can give rise to significant background interferences,decreasing the overall sensor response. For example, in theglucose-specific monitoring system exemplified herein, the GOx enzyme ishighly specific for glucose. However, the biosensor electrode willoxidize any interfering species present in the extracted sample that isoxidizable at the applied potential, resulting in interference with thehydrogen peroxide signal.

In particular, a number of substances commonly found iniontophoretically extracted body fluids can interfere with the hydrogenperoxide signal. Several of these interfering species, such as ascorbicacid, uric acid, and certain amino acids (e.g., tryptophan and tyrosine)can react with the reactive surface of the biosensor and result insubstantial signal interference.

These major interfering species can be divided into two broad classeswhich will behave differently under an applied electric potential; thatis, they are either charged (ionic) substances, or uncharged(substantially neutral) substances. Ascorbic acid and uric acid arenegatively charged (anionic) substances. Tryptophan and tyrosine aresubstantially neutral substances. During iontophoretic sampling,negatively charged species are preferentially extracted toward theanodal electrode, while positively charged and neutral species arepreferentially extracted toward the cathodal electrode. These and otherinterfering species will also be present in any transdermally extractedsample obtained from a subject.

In another embodiment of the invention, an iontophoretic sampling systemis used to measure the concentration of the analyte in the biologicalsystem. The iontophoretic sampling system is configured to have twocollection reservoirs, and provides for a selective extraction step asfollows. Each reservoir is in contact with a single iontophoreticelectrode, and a direct current is used to establish an electricpotential between the two electrodes during iontophoretic extraction.Thus, the electrode in a first reservoir serves as the iontophoreticanode, and the electrode in the second reservoir serves as theiontophoretic cathode. Only the second (cathodic) collection reservoiralso contains the biosensor electrode. Thus, analyte detection iscarried out with an extracted sample that has a reduced amount ofinterfering species, since negatively charged species (e.g., ascorbicacid and uric acid) are preferentially extracted into the anodiccollection reservoir.

The benefits provided by this selective extraction technique can beenhanced using any one, or a combination of the following optionaltechniques. In one embodiment, selective cathodic extraction is combinedwith a means for obtaining a “blank” signal which can be used tosubtract a portion of the cathodic signal which is derived from thepositively charged is and/or neutral interfering species. Moreparticularly, the above-described two-reservoir iontophoretic samplingdevice is used to carry out cathode-selective detection in a continualmanner. Each reservoir is in contact with a single iontophoreticelectrode, and a direct current is used to establish an electricpotential between the two electrodes during iontophoretic extraction.Both reservoirs also contain a biosensor electrode; however, glucosedetection is only carried out in the cathodic reservoir which containsthe GOx enzyme. A number of measurement cycles are carried out in acontinual or continuous manner over an operational period of about 1–24hours or longer. In order to increase the operational lifetime of thecollection reservoirs and iontophoretic electrodes during this continualor continuous sampling, the polarity of the reservoirs can be alternatedsuch that each reservoir serves as the anode at least during a portionof a measurement cycle (i.e., after each iontophoretic half cycle).

In a preferred embodiment of the above method, a detection cycle iscarried out after each iontophoretic half cycle. For example, eachmeasurement cycle entails the following: an iontophoretic extraction iscarried out for a first phase of the extraction step using the firstcollection reservoir as the cathode; a detection step is then carriedout during a first phase of the sensing step to detect substancesextracted during the first phase of the extraction; an iontophoreticextraction is carried out for a second phase of the extraction stepusing the second-reservoir as the cathode; and a second detection stepis then carried out during a second phase of the sensing step. Onlybiosensor measurements from the cathodes are recorded. However,activation of the biosensor electrodes in the anode reservoirs iseffective to deplete (e.g., purge) the concentration of substances(including interfering species) collected therein by chemical reactionwith the reactive surface of the electrode. In this manner, activationof the biosensor electrode in the anode reservoir helps purge thesampling system and substantially remove residual signal componentstherefrom.

Detection is carried out for a sufficient time to ensure that all of theglucose collected in the cathodic reservoir has been subjected to areaction and is therefore no longer detectable, and the biosensorcurrent generated in the cathode is related to the concentration ofglucose in the subject at the approximate time of sample collection. Inaddition, the duration of each detection step should be sufficient toeliminate or substantially reduce the interfering species preferentiallycollected into the anodic reservoir. As will be understood by theordinarily skilled artisan upon reading the instant disclosure, both theiontophoretic extraction phases (half cycles) and the sensing phases canbe tailored to account for the various diffusion characteristics of boththe analyte (glucose) and the major interfering species in a particularcollection reservoir (e.g., reservoir composition and geometry), as wellas for chemical equilibria associated with the analyte/enzyme system(e.g., mutarotation of the glucose molecule).

After the completion of each measurement cycle, the process is repeatedand subsequent measurements are obtained. If desired, the biosensorelectrodes can be activated before the initial measurement cycle, and inbetween subsequent measurement cycles, and/or in between sensing andextraction phases of a measurement cycle. Activation of the biosensorsis maintained for a sufficient period of time to substantially reduce oreliminate background electrochemical signal and/or residual analyte andinterfering species. The biosensor signal can be monitored during theactivation phase and, after the device has been activated for a suitableperiod of time (as indicated by obtaining a stable signal), themeasurement cycle can be initiated. Optionally, a measurement can betaken from the sensor after it reaches stasis, which measurement canthen be used to establish a baseline background signal value. Thisbackground signal value can be subtracted from an actual cathode signalmeasurement value (which includes both analyte-specific and backgroundcomponents) to obtain a corrected measurement value.

In a related embodiment which uses the above alternatingextraction/detection cycles, an additional purging step can be carriedout during one or more of the measurement cycles, wherein the biosensorelectrodes are activated in between a detection step and before asubsequent extraction step (e.g., in between a first phase of thedetection step and a second phase of the extraction step) and in orderto purge the sampling system of residual signal components (bothanalyte-specific and signal due to interfering species). As describedabove, activation of the biosensor electrode depletes both residualanalyte signal and signal due to residual interfering species bychemical reaction with the reactive surface of the electrode. During thepurge step, the biosensor electrode can be operated at a potential ofabout 0.6V or greater in order to ensure that the purge is substantiallycomplete.

In one preferred embodiment of the method, the iontophoretic samplingsystem is operated to provide a measurement cycle having the followingcomponents: a first phase of the extraction step wherein the firstreservoir is operated as the iontophoretic cathode; a first phase of thesensing step wherein an electrochemical signal is obtained from thefirst reservoir; a purging step to remove residual signal from thesampling system; a second phase of the extraction step wherein thesecond reservoir is operated as the iontophoretic cathode; and a secondphase of the sensing step wherein an electrochemical signal is obtainedfrom the second reservoir. If desired, the first reservoir can have theenzyme excluded therefrom, such that the first signal predominantlycontains signal components due to interfering species and backgroundvariations, and the second signal contains analyte-specific signalcomponents. In this manner, a subtraction step can also be used toobtain a signal which is specifically related to the analyte, forexample where the first signal is subtracted from the second signal.

Alternatively, both reservoirs can contain the enzyme such that cathodicsignals can be obtained to provide two “active” analyte-specificsignals. For continuous or continual monitoring applications, the purgestep can performed once, or several times during the continual orcontinuous monitoring period. In one embodiment, the purge step isperformed between each sensing step and a subsequent extraction step. Aswill be understood by the ordinarily skilled artisan upon reading theinstant specification, the preferential extraction techniques describedherein which entail sensing extracted substances in the cathode toobtain analyte-specific information can be used to monitor anypositively charged or neutral substance. If, however, the methods of theinvention are used to monitor negatively charged substances, detectionwill be carried out at the anode, since such analytes will bepreferentially extracted into the anode during iontophoretic extraction.

In another related embodiment which uses the above alternatingextraction/detection cycles, a delay period can also be used to maximizethe detectable glucose signal. More particularly, a delay period isimposed between each iontophoretic half cycle extraction and itscorresponding sensing step. During this delay period, the glucoseanalyte is converted to-the hydrogen peroxide signal, and the effect ofthe rate-limiting process related to mutarotation of glucose is reduced.The delay can be for any suitable period of time, for example, fromabout 15 seconds to about 20 minutes, preferably about 1–7 minutes. Useof such a sensor delay allows for a reduction in biosensor detectiontime, where detection can be limited from about 10 seconds to about 30minutes, preferably from about 30 seconds to about 10 minutes, and mostpreferably from about 90 seconds to about 5 minutes. The combination ofthe sensor delay and the shortened detection time reduces the overallnoise obtained with the biosensor measurement, but provides for the sameanalyte signal, improving the signal-to-noise ratio of the analytemeasurement. As described above, the biosensor electrode can optionallybe kept on for a longer period (e.g., during a purging step) in order tosubstantially deplete signal due to residual analyte and/or interferingspecies in the collection reservoirs.

In yet a further embodiment, preferential (selective cathodic)extraction is combined with a means for reducing the signal produced bythe interfering tryptophan and tyrosine species collected in thecathodic reservoir. In this regard, the iontophoretic sampling systemsdescribed herein typically employ a biosensor having aplatinum-containing electrode which is highly sensitive to the hydrogenperoxide signal produced by the GOx enzyme. The potential of suchbiosensor electrodes is generally set at about 0.6V, where the hydrogenperoxide current-voltage curve is at a plateau. However, at thisoperating potential, the signal from tryptophan and tyrosine cannot bedifferentiated from the hydrogen peroxide (glucose-specific) signal.

Accordingly, in the present embodiment, the potential of the biosensorelectrode is reduced in order to decrease the non-glucose signal. Inparticular, the potential can be reduced to about 0.5V or less, at whichpotentials the signal from tryptophan and tyrosine is greatly reducedwithout a concomitant reduction in the analyte-specific (hydrogenperoxide) signal. In one particular method, the potential is reduced toabout 0.42V in order to substantially eliminate the signal component dueto the tryptophan and tyrosine interfering species.

Each of the techniques used in the above-described two-reservoiriontophoretic sampling devices can be applied to sampling devicescomprising three or more such reservoirs. Accordingly, in yet anotherembodiment of the invention, a method for measuring the concentration ofan analyte present in a biological system is provided which entailstransdermally extracting the analyte from the biological system in anextraction step using an iontophoretic sampling system that comprisesfirst and second iontophoretic collection reservoirs each of which arein operative contact with an iontophoretic electrode. When placed incontact with a skin or mucosal surface, the iontophoretic samplingsystem extracts substances including the analyte into the first andsecond collection reservoirs. The first iontophoretic electrode isoperated as a cathode during the extraction step. The sampling systemfurther comprises a means for passively collecting substances whichdiffuse from, or are secreted by the subject. Passively extractedsubstances are collected into a third collection reservoir that is inoperative contact with a skin or mucosal surface of the subject.Suitable passive collection systems are known in the art, for exampleskin patches which collect perspiration (with or without the use of acholinergic agent or permeation enhancing agent), and the like. Theanalyte which is extracted into the first collection reservoir iscontacted with a sensor in a sensing step to obtain an active signal,and the substances collected into the third collection reservoir arecontacted with the sensor to obtain a blank signal. An analyte-specificsignal is then obtained by subtracting the blank signal from the activesignal. If a negatively charged analyte is being monitored, the firstiontophoretic electrode is operated as an anode during the extractionstep.

In a related embodiment, the analyte is extracted using an iontophoreticsampling system that comprises first, second, and third collectionreservoirs which are respectively in operative contact with first,second, and third iontophoretic electrodes. When these reservoirs arecontacted with a skin or mucosal surface of a subject, the samplingmeans is used to extract substances including the analyte from thebiological system into the first, second, and third collectionreservoirs. The first and second iontophoretic electrodes are operatedas cathodes during the extraction step (i.e., the iontophoretic currentis is divided equally between the cathodes). Sensing is carried out atthe cathodes only, wherein the analyte that is extracted into the firstcollection reservoir is contacted with a sensor to obtain an activesignal and the substances extracted into the second collection reservoirare contacted with the sensor to obtain a blank signal. The blank signalis then subtracted from the active signal to provide an analyte-specificsignal. If desired, the first collection reservoir can contain ananalyte-specific enzyme (e.g., glucose oxidase if glucose is beingmonitored) to provide for the active signal, and the second reservoircan be substantially identical except that the enzyme is excludedtherefrom in order to obtain the blank signal. Alternatively, both thefirst and second collection reservoirs can be used to obtain activesignals (e.g., where both contain an analyte-specific enzyme) in orderto obtain multiple analyte-specific signals. Furthermore, the first andsecond iontophoretic electrodes can be operated as anodes if the analyteof interest is negatively charged.

In a still further embodiment, an iontophoretic sampling system isprovided which can combine cathode-selective measurement with bothactive-blank subtraction and alternating polarity techniques to providefor a substantial reduction in the effect of interfering species onsensor sensitivity. More particularly, a two-reservoir collection systemis used, wherein the collection reservoirs are prepared as anactive/blank system and have identical components (iontophoreticelectrode, biosensor electrode) except that one reservoir contains theGOx enzyme (the active reservoir), while the other does not contain theGOx enzyme (the blank reservoir). These two collection reservoirs areoperated at alternating polarity during iontophoretic extraction.

A microprocessor is used to control the extraction and sensingoperations of the device to provide a measurement cycle which can becarried out in at least a four step protocol as follows. A first phaseof the extraction step is used to generate the blank signal. During thisphase, the blank reservoir is used as the iontophoretic cathode, andglucose (along with positively charged and neutral interfering species)is preferentially collected into the blank reservoir. The negativelycharged interfering species are preferentially collected into the activereservoir which is operated as the iontophoretic anode. After a suitableextraction time, the biosensor electrode in the blank reservoir is usedto obtain a blank signal. A purge step is then carried out, wherein theactive/blank biosensor electrodes are activated to consume residualglucose and/or residual interfering species, particularly the negativelycharged interfering species that were preferentially collected into theactive reservoir during anodic extraction. The polarity of theactive/blank reservoirs is then switched, and cathodic collection iscarried out in the active reservoir. After this second extraction, theglucose analyte is detected in the active reservoir to provide theactive signal. If desired, a sensor delay can also be used between thecathodic extraction and cathodic detection steps of the collection andanalysis phase in order to maximize the glucose signal. Of course thesesteps can be reversed, such as wherein it is desired to obtain theactive signal first during the measurement cycle. Additional, a twoactive system (both reservoirs contain the GOx enzyme) can be used toobtain multiple active signals. Any of the above sampling systems can beoperated continuously or continually, wherein additional purge steps areused to clear the reservoirs between sensing steps and subsequentextraction steps. Further, the methods of the invention (e.g., purgingat an increased potential, detection at a reduced potential, and thelike) can be used to further reduce the affect of interfering species.

Each of the above-described iontophoretic sampling system configurationsand measurement techniques can be used alone, or in any combination, inorder to reduce the effect of interfering species on biosensorsensitivity. These non-invasive glucose monitoring systems allow one tomeasure changes in analyte levels in a subject over a wide range ofanalyte concentrations. Further, the sampling systems can be contactedwith the subject continuously, and automatically obtain samples in orderto measure analyte concentration at preprogrammed intervals. Othermodifications and additions to the above embodiments will be apparent tothose skilled in the art upon reading the present enabling disclosure.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the devices, methods, and formulae of the presentinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Selective Extraction to Reduce Interfering Species

In order to confirm that certain interfering species are preferentiallycollected at the anode of an anode/cathode collection system, thefollowing study was carried out. Iontophoretic sampling was performed ona subject using a prototype iontophoretic sampling system. Theiontophoretic sampling system has two iontophoretic collectionreservoirs each having an iontophoretic electrode.

The subject ingested 250 mg of ascorbic acid at the following timepoints: (1) two hours prior to start of sample collection; (2) one hourprior to start of sample collection; and (3) at the time when samplecollection was first initiated. Iontophoretic sampling (reverseiontophoresis) was performed for 15 minute extraction phases, which werefollowed by a 5 minute period during which buffer sample was removedfrom the iontophoretic collection chambers and replaced with freshbuffer. Samples were taken every 20 minutes for two hours. Theiontophoresis current was run in a DC mode such that the iontophoreticanode and cathode were the same collection reservoir for each sample.Samples were analyzed immediately after collection using HPLC. Theresults of the study are reported below in Table 1, wherein theconcentration of ascorbic acid, uric acid, tyrosine and tryptophan inboth the anode and cathode reservoirs are provided for each of the sixcollection time points.

TABLE 1 Ascorbic Sample Acid Uric Acid Tyrosine Tryptophan A1 5.19472.8066 4.0392 0.3481 A2 2.5519 2.8623 1.7218 0.1096 A3 2.0226 2.6321.1308 0.0726 A4 1.7966 2.5704 1.1026 0.0766 A5 1.7094 2.5064 1.11840.0617 A6 1.6663 2.4746 1.349 0.067 C1 nd 0.392 6.2655 0.3161 C2 nd0.0776 2.8673 0.1213 C3 nd 0.0311 3.2719 0.0954 C4 nd 0.0175 2.53160.1017 C5 nd 0.0215 2.2356 0.0932 C6 nd 0.0106 2.148 0.0838 * nd = notdetermined.As can be seen in Table 1, ascorbic acid is collected solely at theanode (the ascorbic acid level in the cathode samples is undetectable inall samples). In like manner, uric acid is collected predominantly atthe anode, although a low level of uric acid was detectable at thecathode. The anode-to-cathode ratio of ascorbic and uric acid collectionis quite high, varying from about 7.16 to 233.45 to 1. Tyrosine andtryptophan are collected at substantially equal concentrations at theanode and cathode. However, the average flux at the cathode is greaterthan at the anode, as expected for a neutral species.

Example 2 Reduced Sensitivity to Interfering Species at Lower BiosensorOperating Potentials

In order to confirm that certain reduction in biosensor operatingpotential can reduce sensitivity to interfering species, the followingstudy was carried out. Iontophoretic sampling was again performed usinga prototype iontophoretic sampling system. Comparisons were carried outbetween signals obtained using a platinum biosensor electrode operatedat either 0.6V or 0.5V. More particularly, signals for hydrogenperoxide, tyrosine and tryptophan were obtained at each operatingpotential. The results of the study are reported below in Table 2. Theslope is for the linear relationship between detected current at 30seconds, and the concentration of the reacting species.

TABLE 2 30 sec 30 sec Species slope @ 0.6 V slope @ 0.5 V Hydrogen 112.9115 Peroxide Tyrosine 33.9 3.28 Tryptophan 15.23 0.39As can be seen, operating the Pt electrode at a lower potential (0.5V)substantially reduces sensor signals arising from the interferingspecies (tyrosine and tryptophan), while the analyte-specific sensorsignal (hydrogen peroxide) remains unaffected.

1. A monitoring system for measuring an analyte present in one or moresamples in a biological system, said monitoring system comprising: asampling device for providing one or more samples comprising saidanalyte, wherein said sampling device comprises one or moremicro-needles; a sensing device comprising a sensing electrode, whereinsaid sensing electrode obtains a measurement signal from the analyte ineach of said samples, and said measurement signal is specificallyrelated to the analyte amount or concentration in the biological system;and one or more microprocessors in operative communication with thesensing device, wherein said one or more microprocessors compriseprogramming to control (i) operating said sensing device to provide saidmeasurement signals, and (ii) applying one or more processes forselectively favoring analyte-specific signal components over signalcomponents due to interfering species, said processes selected from thegroup consisting of (a) a selective electrochemical detection processthat is performed during said obtaining, said process comprisingcontacting each of said samples with an electrochemical sensingelectrode that is maintained at a potential less than about 0.6V, (b) apurge step that is performed before and/or after said obtaining, whereinsaid step substantially removes residual signal components from thesensing electrode, and (c) any combinations thereof.
 2. The monitoringsystem of claim 1, wherein said one or more microprocessors are inoperative communication with said sampling device and said one or moremicroprocessors further comprise programming to control operating saidsampling device.
 3. The monitoring system of claim 1, said samplingdevice further comprising one or more collection reservoirs.
 4. Themonitoring system of claim 3, wherein a sensing electrode is inoperative contact with at least one of said one or more collectionreservoirs.
 5. The monitoring system of claim 3, wherein at least one ofsaid one or more collection reservoirs comprises an ionically conductivematerial.
 6. The monitoring system of claim 5, wherein said ionicallyconductive material further comprises an enzyme.
 7. The monitoringsystem of claim 3, wherein at least one of said one or more collectionreservoirs comprises a hydrogel.
 8. The monitoring system of claim 7,wherein said hydrogel further comprises an enzyme.
 9. The monitoringsystem of claim 1, wherein said sampling device further uses a samplingtechnique selected from the group consisting of iontophoresis,sonophoresis, suction, electroporation, thermal poration, passivediffusion, and use of laser devices.
 10. The monitoring system of claim1, wherein the analyte is glucose.
 11. The monitoring system of claim 2,wherein said sampling device uses a sampling technique selected from thegroup consisting of iontophoresis, sonophoresis, suction,electroporation, thermal poration, passive diffusion, and use of laserdevices.
 12. The monitoring system of claim 2, wherein the analyte isglucose.
 13. The monitoring system of claim 1, wherein said sensingelectrode comprises a reactive surface comprising a platinum-groupmetal.
 14. The monitoring system of claim 13, wherein saidplatinum-group metal is selected from the group consisting of platinum,palladium, rhodium, ruthenium, osmium, iridium, and combinationsthereof.
 15. The monitoring system of claim 13, wherein said sensingdevice further comprises an enzyme that reacts with the analyte toprovide a chemical signal that is converted at said reactive surface toan electrical signal, wherein said electrical signal is related toanalyte amount or concentration in the biological system.
 16. Themonitoring system of claim 15, wherein said chemical signal is hydrogenperoxide.
 17. The monitoring system of claim 16, wherein saidelectrochemical sensing electrode is maintained at a potential of lessthan about 0.5 volts.
 18. The monitoring system of claim 17, whereinsaid electrochemical sensing electrode is maintained at a potential ofless than about 0.42 volts.
 19. The monitoring system of claim 1,wherein said biological system is a mammal.
 20. The monitoring system ofclaim 19, wherein said mammal is a human.